The long term objective of our laboratory is to understand the mechanism of genetic transformation in H. influenzae at a molecular and biochemical level. In particular, we want to understand the machinery of donor DNA transport through the cell membranes and the complex regulatory pathways that control competence induction. It is toward the latter objective that this grant proposal is directed. In the course of isolating a number of transformation mutants using the mini-Tn10kan transposon mutagenesis approach, we discovered a cluster of mutations spanning a 4-kb region of the chromosome. We recently sequenced this region and flanking sequences and found six tandemly arranged genes which are coordinately induced during competence development. Our studies will focus first on determining whether these genes are cotranscribed as in an operon, or are individually transcribed. In addition, we will identify and study the regulatory genes and cis DNA sequences involved in the transcriptional regulation of the locus. Our specific aims and methods can be summarized as follows: (i) Highest priority will be given to determining the mode of transcriptional expression using plasmid-complementation and mRNA mapping. The 5' and 3' ends of transcripts will be precisely located using S1-nuclease protection assays and primer extension mapping. (ii) Next we will determine which of the six genes are required for transformation and check other genes in the sequenced region for their possible involvement using insertional or site- directed mutagenesis. (iii) Having defined the mode of transcriptional expression, we will seek to identify regulatory DNA sequences in front of the genes using a reporter gene assay. Specifically, we will clone the 5' regions preceding each gene upstream of a promoterless chloramphenicol acetyltransferase (CAT) gene and assay for its activity in response to competence induction. The 5' regions containing regulatory sites will be further analyzed by deletion analysis and site-directed mutagenesis to locate and identify the sites. (iv) CAT activity will also be determined in strains mutant for the known competence regulatory genes crp and sxy1. (v) We will investigate whether the product of comA, the first gene in the locus, regulates its own expression, and that of the other genes, in strains containing a comA-lacZ operon or protein fusion in tandem with an intact comA gene. (vi) We will search for other regulatory genes affecting the expression of the locus. (vii) Finally, we will search for competence-specific sigma factors that might be involved in global switching from constitutive-gene to competence gene expression during competence induction. For this purpose, we will examine protein extracts from competent and non-competent cells using anti-sigma antibodies in Western blots. An alternate approach will employ PCR-amplification using sigma factor-specific degenerate primers for highly conserved regions.